Response of Chrysantemum Plant to Addition of Broiler Manure as a Substitute for Commercial Substrate

Dos tipos diferentes de pollinaza mezclados con el material de cama (paja o serrín) añadidas en tasas de 0 y 10% más dos substratos comerciales de turba (turba negra y turba rubia) se usaron para estudiar el crecimiento de crisantemo en maceta. En todos los casos, la calidad y el tamaño de las plantas fue mejor con las mezclas de substratos de turba negra que con las mezclas de turba rubia. Con las mezclas de turba negra con pollinaza hubo un aumento significativo del número de flores sin que hubiese un aumento en el tamaño de las plantas. También se pudo observar que con la adición de pollinaza, el substrato de turba rubia mejoró significativamente los parámetros de crecimiento estudiados, esto fue debido a que el pH del substrato fue más adecuado para el cultivo. Por último, no hubo mortalidad de plantas con las mezclas experimentadas, pero cuando la cantidad de gallinaza aumentó en la mezcla fue más notable la mortalidad de plantas.

The effect of a mono component protease and different AMEn levels in broiler diets on growth performance from 1 to 18 days of age.

The objective of the present study was to evaluate the effect of the inclusion of a mono component serine protease (RONOZYME ProAct, DSM Nutritional Products) in diets with two different AMEn contents on apparent ileal digestibility (AID) of amino acids (AA) and growth performance in broilers from 1 to 18 days of age.

Efecto de la edad y el nivel de vitamina E en dietas de pollos broiler sobre la modulación de la respuesta inmune.

La vitamina E pertenece al grupo de nutrientes con capacidad reguladora del sistema inmune (Koutsos y Klasing, 2008). En aves, la vitamina E induce cambios tanto en el sistema inmune innato como en el específico, mejora la función fagocítica de los macrófagos, amortigua la respuesta en fase aguda, disminuye la proporción de heterófilos y potencia la síntesis de anticuerpos (Koutsos y Klasing, 2008; Khan et al., 2012). Recientemente se ha demostrado que parte del efecto de la vitamina E está ligado a su capacidad de actuar directamente sobre factores de transcripción nuclear que modulan la expresión de citoquinas, tales como el factor-Kappa B o el PPAR γ (Koutsos y Klasing, 2008; Nakamura y Omaye, 2009). Por otro lado, el impacto de la vitamina E sobre la modulación del sistema inmune viene definido por factores tales como la edad y la relación dosis-respuesta. En broiler la inclusión en la dieta de niveles moderadamente superiores (25-50 UI/kg dieta) al recomendado por el National Research Council (NRC, 1994; 10 UI/kg) aumenta los títulos de anticuerpos tras la vacunación (Friedman et al., 1998; Leshchinsky y Klasing, 2001; Lin y Chang, 2006). Sin embargo, niveles superiores a 150 UI/kg tienen un efecto supresor sobre la producción de anticuerpos (Koutsos y Klasing, 2008). En relación a la edad es importante determinar aquellos periodos o “ventanas” en los que es necesario estimular el sistema inmune (Kogut, 2009). Un periodo a considerar sería el de las dos primeras semanas de vida del pollo debido a la menor funcionalidad de macrófagos y heterófilos (Kogut, 2009). El presente trabajo tiene como objetivo evaluar el efecto del nivel de Vitamina E y la edad sobre la modulación de la respuesta inmune en pollos. Para ello se estudió el efecto de la inclusión de 40 y 160 ppm de vitamina E en la dieta sobre la expresión de la interleuquina 2 (IL 2), el interferón gamma (IFN γ) y el factor de crecimiento mielomonocítico (MGF) en el bazo de los animales a los 7 y 21 días de edad.

Role of fibre in broiler diets - Firend or foe?

The fibrous fraction of the feeds encompasses a group of heterogeneous compounds differing in chemical composition and physical properties (Graham and Aman, 1991, Bach Knudsen, 2001). Dietary fiber is the most used term to define the fiber fraction of ingredients and feeds, and includes cell walls, stored non-starch polysaccharides (NSP), and lignin (Bach Knudsen, 2001). Based on their physico-chemical properties, DF can be divided into soluble and insoluble fractions with distinct effects on digestive physiology and animal metabolism. Consequently, the benefits of fiber inclusion in poultry diets will vary depending on factors such as characteristics of the fiber source, type of bird, and digestive health status.

Positional enhancer-blocking activity of the chicken β-globin insulator in transiently transfected cells

It is thought that insulators demarcate transcriptionally and structurally independent chromatin domains. Insulators are detected by their ability to block enhancer–promoter interactions in a directional manner, and protect a transgene from position effects. Most studies are performed in stably transformed cells or organisms. Here we analyze the enhancer-blocking activity of the chicken β-globin insulator in transient transfection experiments in both erythroid and nonerythroid cell lines. We show that four tandem copies of a 90-bp fragment of this insulator were able to block an enhancer in these experiments. In circular plasmids, placement on either side of the enhancer reduced activity, but when the plasmid was linearized, the enhancer-blocking activity was observed only when the insulator was placed between the promoter and the enhancer. These observations are consistent with the position-dependent enhancer-blocking activity of the insulator observed in stable transformation experiments.

Chicken Erythroid AE1 Anion Exchangers Associate with the Cytoskeleton During Recycling to the Golgi

Chicken erythroid AE1 anion exchangers receive endoglycosidase F (endo F)-sensitive sugar modifications in their initial transit through the secretory pathway. After delivery to the plasma membrane, anion exchangers are internalized and recycled to the Golgi where they acquire additional N-linked modifications that are resistant to endo F. During recycling, some of the anion exchangers become detergent insoluble. The acquisition of detergent insolubility correlates with the association of the anion exchanger with cytoskeletal ankyrin. Reagents that inhibit different steps in the endocytic pathway, including 0.4 M sucrose, ammonium chloride, and brefeldin A, block the acquisition of endo F-resistant sugars and the acquisition of detergent insolubility by newly synthesized anion exchangers. The inhibitory effects of ammonium chloride on anion exchanger processing are rapidly reversible. Furthermore, AE1 anion exchangers become detergent insoluble more rapidly than they acquire endo F-resistant modifications in cells recovering from an ammonium chloride block. This suggests that the cytoskeletal association of the recycling anion exchangers occurs after release from the compartment where they accumulate due to ammonium chloride treatment, and prior to their transit through the Golgi. The recycling pool of newly synthesized anion exchangers is reflected in the steady-state distribution of the polypeptide. In addition to plasma membrane staining, anion exchanger antibodies stain a perinuclear compartment in erythroid cells. This perinuclear AE1-containing compartment is also stained by ankyrin antibodies and partially overlaps the membrane compartment stained by NBD C6-ceramide, a Golgi marker. Detergent extraction of erythroid cells in situ has suggested that a substantial fraction of the perinuclear pool of AE1 is cytoskeletal associated. The demonstration that erythroid anion exchangers interact with elements of the cytoskeleton during recycling to the Golgi suggests the cytoskeleton may be involved in the post-Golgi trafficking of this membrane transporter.

5-Methylcytosine-DNA glycosylase activity is present in a cloned G/T mismatch DNA glycosylase associated with the chicken embryo DNA demethylation complex

We previously have shown that DNA demethylation by chicken embryo 5-methylcytosine DNA glycosylase (5-MCDG) needs both RNA and proteins. One of these proteins is a RNA helicase. Further peptides were sequenced, and three of them are identical to the mammalian G/T mismatch DNA glycosylase. A 3,233-bp cDNA coding for the chicken homologue of human G/T mismatch DNA glycosylase was isolated and sequenced. The derived amino acid sequence (408 aa) shows 80% identity with the human G/T mismatch DNA glycosylase, and both the C and N-terminal parts have about 50% identity. As for the highly purified chicken embryo DNA demethylation complex the recombinant protein expressed in Escherichia coli has both G/T mismatch and 5-MCDG activities. The recombinant protein has the same substrate specificity as the chicken embryo 5-MCDG where hemimethylated DNA is a better substrate than symmetrically methylated CpGs. The activity ratio of G/T mismatch and 5-MCDG is about 30:1 for the recombinant protein expressed in E. coli and 3:1 for the purified enzyme from chicken embryos. The incubation of a recombinant CpG-rich RNA isolated from the purified DNA demethylation complex with the recombinant enzyme strongly inhibits G/T mismatch glycosylase while slightly stimulating the activity of 5-MCDG. Deletion mutations indicate that G/T mismatch and 5-MCDG activities share the same areas of the N- and C-terminal parts of the protein. In reconstitution experiments RNA helicase in the presence of recombinant RNA and ATP potentiates the activity of 5-MCDG.

Isolation and characterization of the chicken m2 acetylcholine receptor promoter region: Induction of gene transcription by leukemia inhibitory factor and ciliary neurotrophic factor

We have isolated the promoter region and determined the start sites of transcription for the gene encoding the chicken m2 (cm2) muscarinic acetylcholine receptor. Transfection experiments, using cm2-luciferase reporter gene constructs, demonstrated that a 789-bp genomic fragment was sufficient to drive high level expression in chicken heart primary cultures, while an additional 1.2-kb region was required for maximal expression in mouse septal/neuroblastoma (SN56) cells. Treatment of SN56 cells with the cytokines ciliary neurotrophic factor and leukemia inhibitory factor increases expression of endogenous muscarinic acetylcholine receptors and results in a 4- to 6-fold induction of cm2 promoter driven luciferase expression. We have mapped a region of the cm2 promoter that is necessary for induction by cytokines.

Ectopic bone morphogenetic proteins 5 and 4 in the chicken forebrain lead to cyclopia and holoprosencephaly

Proper dorsal–ventral patterning in the developing central nervous system requires signals from both the dorsal and ventral portions of the neural tube. Data from multiple studies have demonstrated that bone morphogenetic proteins (BMPs) and Sonic hedgehog protein are secreted factors that regulate dorsal and ventral specification, respectively, within the caudal neural tube. In the developing rostral central nervous system Sonic hedgehog protein also participates in ventral regionalization; however, the roles of BMPs in the developing brain are less clear. We hypothesized that BMPs also play a role in dorsal specification of the vertebrate forebrain. To test our hypothesis we implanted beads soaked in recombinant BMP5 or BMP4 into the neural tube of the chicken forebrain. Experimental embryos showed a loss of the basal telencephalon that resulted in holoprosencephaly (a single cerebral hemisphere), cyclopia (a single midline eye), and loss of ventral midline structures. In situ hybridization using a panel of probes to genes expressed in the dorsal and ventral forebrain revealed the loss of ventral markers with the maintenance of dorsal markers. Furthermore, we found that the loss of the basal telencephalon was the result of excessive cell death and not a change in cell fates. These data provide evidence that BMP signaling participates in dorsal–ventral patterning of the developing brain in vivo, and disturbances in dorsal–ventral signaling result in specific malformations of the forebrain.

CXR, a chicken xenobiotic-sensing orphan nuclear receptor, is related to both mammalian pregnane X receptor (PXR) and constitutive androstane receptor (CAR)

Nuclear receptors constitute a large family of ligand-modulated transcription factors that mediate cellular responses to small lipophilic molecules, including steroids, retinoids, fatty acids, and exogenous ligands. Orphan nuclear receptors with no known endogenous ligands have been discovered to regulate drug-mediated induction of cytochromes P450 (CYP), the major drug-metabolizing enzymes. Here, we report the cloning of an orphan nuclear receptor from chicken, termed chicken xenobiotic receptor (CXR), that is closely related to two mammalian xenobiotic-activated receptors, the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). Expression of CXR is restricted to tissues where drug induction of CYPs predominantly occurs, namely liver, kidney, small intestine, and colon. Furthermore, CXR binds to a previously identified phenobarbital-responsive enhancer unit (PBRU) in the 5′-flanking region of the chicken CYP2H1 gene. A variety of drugs, steroids, and chemicals activate CXR in CV-1 monkey cell transactivation assays. The same agents induce PBRU-dependent reporter gene expression and CYP2H1 transcription in a chicken hepatoma cell line. These results provide convincing evidence for a major role of CXR in the regulation of CYP2H1 and add a member to the family of xenobiotic-activated orphan nuclear receptors.

Segment-specific pattern of sympathetic preganglionic projections in the chicken embryo spinal cord is altered by retinoids

Sympathetic preganglionic neurons exhibit segment-specific projections. Preganglionic neurons located in rostral spinal segments project rostrally within the sympathetic chain, those located in caudal spinal segments project caudally, and those in midthoracic segments project either rostrally or caudally in segmentally graded proportions. Moreover, rostrally and caudally projecting preganglionic neurons are skewed toward the rostral and caudal regions, respectively, of each midthoracic segment. The mechanisms that establish these segment-specific projections are unknown. Here we show that experimental manipulation of retinoid signaling in the chicken embryo alters the segment-specific pattern of sympathetic preganglionic projections and that this effect is mediated by the somitic mesoderm. Application of exogenous retinoic acid to a single rostral thoracic somite decreases the number of rostrally projecting preganglionic neurons at that level. Conversely, disrupting endogenous synthesis of retinoic acid in a single caudal thoracic somite increases the number of rostrally projecting preganglionic neurons at that level. The number of caudally projecting neurons does not change in either case, indicating that the effect is specific for rostrally projecting preganglionic neurons. These results indicate that the sizes of the rostrally and caudally projecting populations may be independently regulated by different factors. Opposing gradients of such factors along the longitudinal axis of the thoracic region of the embryo could be sufficient, in combination, to determine the segment-specific identity of preganglionic projections.

Leucine zipper motif of chicken histone acetyltransferase-1 is essential for in vivo and in vitro interactions with the p48 subunit of chicken chromatin assembly factor-1

We cloned cDNA encoding chicken cytoplasmic histone acetyltransferase-1, chHAT-1, comprising 408 amino acids including a putative initiation Met. It exhibits 80.4% identity to the human homolog and possesses a typical leucine zipper motif. The glutathione S-transferase (GST) pull-down assay, involving truncated and missense mutants of the chicken chromatin assembly factor-1 (chCAF-1)p48, revealed not only that a region (comprising amino acids 376–405 of chCAF-1p48 and containing the seventh WD dipeptide motif) binds to chHAT-1 in vitro, but also that mutation of the motif has no influence on the in vitro interaction. The GST pull-down assay, involving truncated and missense chHAT-1 mutants, established that a region, comprising amino acids 380–408 of chHAT-1 and containing the leucine zipper motif, is required for its in vitro interaction with chCAF-1p48. In addition, mutation of each of four Leu residues in the leucine zipper motif prevents the in vitro interaction. The yeast two-hybrid assay revealed that all four Leu residues within the leucine zipper motif of chHAT-1 are necessary for its in vivo interaction with chCAF-1p48. These results indicate not only that the proper leucine zipper motif of chHAT-1 is essential for its interaction with chCAF-1p48, but also that the propeller structure of chCAF-1p48 expected to act as a platform for protein–protein interactions may not be necessary for this interaction of chHAT-1.

Light-induced phase-delay of the chicken pineal circadian clock is associated with the induction of cE4bp4, a potential transcriptional repressor of cPer2 gene

The chicken pineal gland contains the autonomous circadian oscillator together with the photic-input pathway. We searched for chicken pineal genes that are induced by light in a time-of-day-dependent manner, and isolated chicken homolog of bZIP transcription factor E4bp4 (cE4bp4) showing high similarity to vrille, one of the Drosophila clock genes. cE4bp4 was expressed rhythmically in the pineal gland with a peak at very early (subjective) night under both 12-h light/12-h dark cycle and constant dark conditions, and the phase was nearly opposite to the expression rhythm of cPer2, a chicken pineal clock gene. Luciferase reporter gene assays showed that cE4BP4 represses cPer2 promoter through a E4BP4-recognition sequence present in the 5′-flanking region, indicating that cE4BP4 can down-regulate the chick pineal cPer2 expression. In vivo light-perturbation studies showed that the prolongation of the light period to early subjective night maintained the high level expression of the pineal cE4bp4, and presumably as a consequence delayed the onset of the induction of the pineal cPer2 expression in the next morning. These light-dependent changes in the mRNA levels of the pineal cE4bp4 and cPer2 were followed by a phase-delay of the subsequent cycles of cE4bp4/cPer2 expression, suggesting that cE4BP4 plays an important role in the phase-delaying process as a light-dependent suppressor of cPer2 gene.

Structure of the catalytic fragment of poly(AD-ribose) polymerase from chicken.

The crystal structures of the catalytic fragment of chicken poly(ADP-ribose) polymerase [NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] with and without a nicotinamide-analogue inhibitor have been elucidated. Because this enzyme is involved in the regulation of DNA repair, its inhibitors are of interest for cancer therapy. The inhibitor shows the nicotinamide site and also suggests the adenosine site. The enzyme is structurally related to bacterial ADP-ribosylating toxins but contains an additional alpha-helical domain that is suggested to relay the activation signal issued on binding to damaged DNA.

Assignment of Rfp-Y to the chicken major histocompatibility complex/NOR microchromosome and evidence for high-frequency recombination associated with the nucleolar organizer region.

Rfp-Y is a second region in the genome of the chicken containing major histocompatibility complex (MHC) class I and II genes. Haplotypes of Rfp-Y assort independently from haplotypes of the B system, a region known to function as a MHC and to be located on chromosome 16 (a microchromosome) with the single nucleolar organizer region (NOR) in the chicken genome. Linkage mapping with reference populations failed to reveal the location of Rfp-Y, leaving Rfp-Y unlinked in a map containing >400 markers. A possible location of Rfp-Y became apparent in studies of chickens trisomic for chromosome 16 when it was noted that the intensity of restriction fragments associated with Rfp-Y increased with increasing copy number of chromosome 16. Further evidence that Rfp-Y might be located on chromosome 16 was obtained when individuals trisomic for chromosome 16 were found to transmit three Rfp-Y haplotypes. Finally, mapping of cosmid cluster III of the molecular map of chicken MHC genes (containing a MHC class II gene and two rRNA genes) to Rfp-Y validated the assignment of Rfp-Y to the MHC/NOR microchromosome. A genetic map can now be drawn for a portion of chicken chromosome 16 with Rfp-Y, encompassing two MHC class I and three MHC class II genes, separated from the B system by a region containing the NOR and exhibiting highly frequent recombination.